Role of glutathione and nucleotide excision repair in modulation of cisplatin activity with O6-benzylguanine

Purpose: Modulation of platinating agent cytotoxicity has important clinical implications as a result of their widespread use in the treatment of many different cancers. O-6-Benzylguanine (BG) enhances the cytotoxicity of cisplatin against several human tumor lines. The purpose of our work was to elucidate whether BG affects pathways prior to DNA damage (i.e., glutathione, GSH) or following DNA damage (i.e., nucleotide excision repair, NER). Methods: In efforts to determine the mechanism of enhancement we: (1) evaluated whether different sequences of BG plus cisplatin treatment differed in their ability to enhance cisplatin-induced cytotoxicity and DNA platination; (2) determined the effect of BG on GSH and glutathione S-transferase (GST) activity and; (3) determined whether BG enhanced cisplatin-induced cytotoxicity in cells lacking specific enzymes in the NER pathway. Colony-forming assay, atomic absorption spectroscopy and HPLC were employed to measure tumor cell growth inhibition, quantitate the amount of platinum on DNA, and determine intracellular GSH concentrations, respectively. Results: Increased cytotoxicity and platination of DNA was observed when cells were exposed to BG prior to and/or during cisplatin treatment and not when BG followed cisplatin treatment. BG did not significantly alter GST activity with minimal depletion of GSH. In contrast, buthionine sulfoximine (BSO) caused a much more dramatic decrease in GSH than BG that was not accompanied by a dramatic increase in sensitivity to cisplatin. Furthermore, BG enhanced the cytotoxicity of cisplatin in a series of cell lines deficient in NER. Conclusions: Overall, our results suggest that the mechanism of enhancement involves neither the GSH nor the NER pathways, but triggers an event prior to DNA platination damage that ultimately results in increased cytotoxicity, apoptosis and increased platination levels.